Project Summary/ Abstract The central hypothesis and theme of the program project is that altered signaling at the level of sarcomeric proteins occurs in the course of compensation to key hemodynamic stressors (pressure and volume) that are significant factors in the decompensations leading to pump failure and death. The approach in Project 2 relates micromechanical signaling to cell hypertrophy by growing cardiac myocytes (CMs) in microenvironments of different compliance (stiffness) to mimic chronic load (disease), or by loading with micromagnets to mimic acute changes. We focus on actin and the actin capping protein, CapZ, to study sarcomere assembly. The specific hypothesis tested is that actin assembly depends on CapZ modification by mechano-transduction signaling pathways, such as deacetylation via HDACs, phosphorylation by PKC?, and phosphatidylinositol 4,5- bisphosphate (PIP2) binding. This proposal presents innovative approaches to provide local forces to CMs in three dimensions and determine cellular changes using state-of-art biophysical and proteomic techniques. Specific Aim 1 determines the effect of chronic load generated by varying stiffness of the microenvironment on sarcomere assembly. Chronic load is altered by interaction of CMs in 3D culture with microstructures of variable stiffness (10 to 400kPa). Contractility is assessed by shortening using line scan kymographs. Actin filament assembly is assessed by fluorescence recovery after photobleaching (FRAP) of CapZ and actin using GFP adenoviral infection. Techniques are refined on neonatal rat ventricular myocytes prior to CMs derived from human induced pluripotent stem cells (hIPSC-CMs), and CMs from human and adult rabbit hearts. Specific Aim 2 determines the effect of dynamic loading delivered to living CMs by micromagnets on contractility, signal translocation and sarcomere assembly. Dynamic forces are changed by novel micromagnets that anchor to CMs and can be loaded by external magnetic fields. Contractility and the dynamics CapZ and actin are compared by FRAP at the subcellular level. Translocation of labeled signaling molecules is tracked after micromagnet loading using time lapse live cell imaging. Specific Aim 3 determines how mechano-feedback signaling modifies CapZ and coordinates actin assembly. To test the hypothesis that the mechanism of thin filament assembly depends on modifications of CapZ, proteomic and biochemical approaches are used on CMs under various loading conditions, and also in failing or LVAD human heart. Coordination of signaling by acetylation, phosphorylation and/or PIP2 binding to CapZ is tested by specific molecular interventions with FRAP and proteomic readouts. Innovation and impact: The well established team of Russell (physiology at UIC) and Desai (bioengineering at UCSF) is uniquely suited to address how sarcomeres in individual myocytes respond to bioengineered microenvironments. The long-term goal is to understand cardiac remodeling, which is almost certain to involve mechano-signaling and may provide a path for novel therapeutic targets to avoid maladaptation over time.